Ultrastructure of Lassa virus

We produced live LASV in African green monkey kidney epithelial cells under BSL-4 containment conditions. Cell supernatants containing LASV virions were subsequently inactivated by chemical cross-linking using 4% paraformaldehyde (PFA). Prior to structural analysis, we optimized a gradient ultracentrifugation based protocol for virion purification (see Materials and Methods).

Tomographic 3D reconstructions (‘tomograms’) calculated from electron cryomicroscopy tilt series of purified virions revealed both the exterior and interior density of LASV virions (Fig 1A). The virions were roughly spherical (S1 Fig, LASV) with diameter of 132±22 nm (S2 Fig, LASV). Consistent with previous observations [15], a density plot calculated normal to the membrane (S3 Fig, LASV) revealed i. an outer layer, comprising the GP spikes, ii. two middle layers, comprising the two leaflets of the lipid bilayer, and iii. two internal layers (‘inner track 1’ and ‘inner track 2’) [15], the first comprising GP intra-viral tails (see below) and the second comprising Z and possibly ribonucleoprotein (RNP), which also partially fills the virion interior (Fig 1A).

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Fig 1. Structure of Lassa virus solved using electron cryotomography and sub-tomogram averaging.

(A) A slice through a 3D tomogram of LASV. Three glycoprotein are spikes indicated (white arrowheads). The inset shows a magnified view of the area indicated (dotted line). The membrane (M), matrix (Z), and density bridging a spike to the Z layer (black arrowhead) are indicated. (B) LASV GP spike structure segmented to three spike protomers (different shades of brown), membrane (light blue), and the Z layer (dark blue). Intra-viral tails connecting the spike to the underlying matrix are indicated (arrowhead). (C) One LASV virion is shown cut open highlighting the irregular organization of the glycoprotein spikes, membrane, Z layer, and RNP density (gray).

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Glycoprotein spike trimers connect to the underlying matrix and are randomly distributed on the virion surface

By averaging ~6,500 volumes (‘sub-tomograms’) of GP spikes extracted from tomograms of 48 inactivated LASV virions, we solved the 3D structure of the GP spike (Fig 1B and 1C and Table 1) to 14 Å resolution, as estimated by the Fourier shell correlation (FSC; S4 Fig). The structure revealed a trimeric assembly, measuring ~9 nm in height and ~10 nm in width (Fig 1B). Three separate densities (‘legs’), spaced by ~4 nm, anchor the spike to the membrane surface. By analogy to the trimeric structures of the HIV-1 [16] and Ebola virus glycoprotein [17] spikes, where the class I fusion glycoproteins subunits are membrane-proximal, we putatively assign these membrane proximal legs to the GP2 glycoprotein. This is supported by the presence of a trans-membrane (TM) region at the C-terminus of the LASV GP2, as predicted by sequence analysis. As SSP interacts with GP2 [8,18], it may also contribute to these leg densities. Earlier biochemical studies have suggested several topologies for SSP [8,19–21]. However, at 14-Å resolution we were unable to resolve the structure of SSP, making structural arrangements and orientation of SSP within the membrane proximal region difficult to predict. Furthermore, at this resolution the TM regions cannot be resolved and were thus not visible in our structure.

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Table 1. Data acquisition and processing statistics.

https://doi.org/10.1371/journal.ppat.1005418.t001

Visual inspection of the tomographic slices revealed connections bridging the spike to the Z layer underneath the membrane (Fig 1A, inset). Density corresponding to these connections was ordered in the averaged GP structure (Fig 1B). The measured volume of this intra-viral density corresponded to molecular mass of ~18 kDa. This mass is consistent with the three 41-residue long intra-viral tails of GP2 (14 kDa in total), indicated by sequence analysis. We thus putatively assign the 18-kDa intra-viral density collectively to the three tails of GP2. In addition, SSP may contribute additional mass to these tails.

Template matching allowed mapping the distribution of spikes on the virions (N = 48). The spikes (273±22) almost fully covered the virion surface (Fig 1C). No higher-order clustering was observed (S5 Fig), confirming the well-accepted notion that LASV is not icosahedrally symmetric and suggesting that no specific interactions between the GP trimers exist.

Structure of the native LASV GP from virus-like particles

To account for possible effects of chemical fixation, we exploited a previously reported stable cell line expressing non-infectious virus-like particles (VLPs) displaying the full-length LASV GP but lacking the Z [22]. Similar to the fixed virions, VLPs purified from cell culture supernatants were covered with GP spikes (S1 Fig; VLP pH 7). However, size measurements (S2 Fig) showed that VLPs were significantly smaller (diameter 82±15 nm) than the virions (132±22 nm) and also their shape was more variable (S1 Fig). This is most likely due to the absence of Z, which modulates virion budding and RNP packaging [23].

Following the same sub-tomogram averaging approach as for the virions, we solved the structure of the native GP spike from the VLPs (Fig 2, pH 7; Table 1) to 14-Å resolution (S4 Fig). Visual inspection revealed that the VLP-derived spike ectodomain structure shared the same tripodal architecture and structural features with the virion-derived spike structure (S6A Fig). To provide a quantitative measure for similarity, we performed cross-FSC analysis, which indicated that the two ectodomains agreed to 16-Å resolution (S6B Fig).

The density profile calculated across the VLP membrane was practically identical to that of the virion for the density corresponding both to the GP ectodomain and GP2 intra-viral tails. As expected, density assigned to Z in the virion was absent in the VLPs that lacked this component (S3 Fig, VLP). Surprisingly, the GP2 intra-viral tails that were ordered in the virions (Fig 1B) were disordered in the VLP-derived GP structure (S6A Fig). While it is possible to attribute the ordering of the intra-viral tails to the GP–Z interaction [22,24], we cannot exclude the possible effect of chemical fixation in ordering the intra-viral tails.

The direct comparison of the virion-derived and VLP-derived GP structures supports our assignment of the density in the first density layer under the membrane (denoted earlier as ‘inner track 1’) [15] to the GP2 intra-viral tails. We note that this assignment differs from a previous assignment, where the ‘inner track 1’ was assigned to the Z layer in the two-dimensional cryo-EM images of three other arenaviruses, namely Pichinde, Tacaribe and lymphocytic choriomeningitis virus [15]. We note, however, that we cannot exclude structural differences between LASV and the other studied arenaviruses. It is also possible that Z contributes some density to the inner track 1 in LASV. In conclusion, by comparison to the VLP system, we could verify the LASV GP spike structure and our assignment of the intra-viral GP2 density.

Glycoprotein spike binding to LAMP1 at endosomal pH

Following virus internalization into the host cell through GP attachment to α-dystroglycan [25], DC-SIGN [26,27], or other cellular receptors [26], fusion of viral and host cell membranes occurs at an unusually low pH of 3–4.5 [28], a process mediated by recognition of the host lysosomal receptor, LAMP1 [11]. Receptor binding is modulated by pH; GP binds to α-dystroglycan at pH 8.0 but not at pH 6.0 and below, and, conversely, LAMP1 binding occurs only at pH 6.0 and below [11].

To study GP conformation at endosomal pH, we determined the VLP-derived GP structure at pH 5.2 (Table 1 and Fig 2, pH 5, and S1 Fig VLP pH 5). The structure was resolved at 16-Å resolution (S4 Fig) and revealed conformational differences when compared to the same structure at pH 7.3 (Fig 2, pH 7). These can be attributed to a conformational difference at the membrane-proximal legs and a slight opening up of the spike, creating crevices at the top of the spike between the GP subunits.

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Fig 2. Structural transitions and LAMP1 binding of the LASV GP spikes upon acidification.

(A) GP structures at different pHs are shown from side (top row) and top (bottom row). All volumes were filtered to 17-Å resolution, rendered at molecular threshold corresponding to the expected molecular mass, and colored as in Fig 1. Residual density corresponding to LAMP1 is colored in green. Inserts in the lower left corners show a close-up of the interface between two spike monomers. Insets in the top right corners show Western blot analysis of GP1 and GP2 subunits. The arrowheads indicate the missing density in the central top part and side of the pH 3 structure.

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To map the LAMP1 binding site on the GP spike, we repeated the electron cryomicroscopy-based structure analysis after addition of purified LAMP1 at pH 5.5 (Fig 2, pH 5 + LAMP1). First we expressed and purified the predicted membrane distal, highly glycosylated β-prism domain of human LAMP1 (residues 27–194) [29]. This domain was biologically active binding to VLPs at pH 5.0 (S7 Fig). The GP–LAMP1 complex structure was resolved to 15-Å resolution (S4 Fig). Additional density attributed to LAMP1 was evident at the top of the spike and located close to the crevices observed in the VLP GP structure in absence of LAMP1 (Fig 2; pH 5 + LAMP1). The volume of LAMP1 density was less than expected based on the molecular mass of the LAMP1 glycoprotein fragment used for analysis (S7 Fig), suggesting that LAMP1 either bound to VLP GP in sub-stoichiometric amounts and/or that the binding was flexible. In conclusion, the location of this additional residual density allowed mapping of the LAMP1 binding site to the membrane distal part of the spike.

GP1 subunit is shed from the spike at lysosomal pH

In line with previous reports [11], our Western blot analysis revealed that the GP1 subunit was shed from the VLPs at pH 3.0 and 4.0 (S8 Fig). To determine the LASV GP structure in the absence of GP1, we performed electron cryotomography and sub-tomogram averaging of VLP-derived GPs at pH 3.0 (Table 1 and S1 Fig, VLP pH 3). The structure was missing density in the membrane-distal part of the spike (Fig 2, pH 3), suggesting that the GP1 occupies this region.

In the absence of a target membrane, the pH 3 structure may constitute SSP in complex with either a non-productive post-fusion conformation of GP2 or an intermediate conformation prior to fusion. Indeed, it is strikingly different from the canonical post-fusion ectodomain structures of other class I fusion proteins, which display a central trimeric alpha-helical coiled coil (S9 Fig) [3,30]. However, we also cannot exclude the possibility that a fraction of GP2 has mediated fusion of some VLPs with one another. The resulting post-fusion conformation species in this fraction may have been excluded from our analysis. Despite these limitations, the pH 3 structure allowed putative localization the GP1 subunit to the membrane distal region of the spike. This is consistent with the mapped LAMP1 binding site in the same region, as LAMP1 binds to the GP1 subunit [10].

Fitting of GP1 structure into the GP spike density

The structure of LASV GP1 has been recently solved by X-ray crystallography [10]. To localize the GP1 and determine its orientation relative to the LAMP1 binding site, we used volumetric correlation to fit the GP1 crystal structure into our GP reconstruction (Fig 3 and S10 Fig). As the GP1 has been crystallized at pH 5 and acidic pH has been suggested to have an effect on GP1 conformation [10], we used our GP structure derived from VLPs at pH 5 for fitting. The fitting was unambiguous (S10C Fig; cross-correlation coefficient 0.86) and placed the GP1 structure to the membrane distal part of the spike (Fig 3). Interestingly, the triad of histidines that has been indicated in LAMP1 binding [10] points towards the crevice between GP1 subunits (Fig 3), the putative LAMP1 binding side (Fig 2). The histidines may act as a pH sensor [10] opening up the spike at pH 5 to create a binding site for LAMP1, but further studies are needed to test this hypothesis.

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Fig 3. Location of the GP1 subunit in the glycoprotein spike.

(A) Crystallographic structure [10](brown ribbon; PDB:4ZJF) was fitted into density (transparent surface) derived by electron cryomicroscopy and subtomogram averaging of GP spikes from virus-like particles at pH 5. The glycans are represented with sticks and colored in green. The histidines indicated in binding of an intracellular receptor (lysosome-associated membrane protein 1, LAMP1) are labeled and colored in red. This triad of histidines faces a crevice (white arrowhead) between the spike subunits. The three-fold symmetry axis is indicated with a black triangle. (B) Same fitting as in A is shown from the side.

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Preparation, inactivation and purification of LASV

All work with infectious LASV was performed under the highest safety precautions in the biosafety level-4 (BSL-4) facility at the Institute of Virology in Marburg. Vero cells were infected with LASV (strain Josiah) at a multiplicity of infection of 0.1. Infected cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco, Thermo Fischer Scientific, Waltham, MA) supplemented with 2% fetal calf serum (FCS; Gibco, Thermo Fischer Scientific, Waltham, MA), penicillin (100 U/ml), streptomycin (100 mg/ml), and L-glutamine (2 mmol/l) (Invitrogen, Thermo Fischer Scientific, Waltham, MA) at 37°C under 5% CO₂. Supernatant was collected 5 days after infection and cleared twice by centrifugation at 5,000 g for 30 min. Supernatant containing LASV was then subjected to virus inactivation using PFA (4% final concentration) in DMEM for 24 h. The sample was removed from the BSL-4 facility and after additional 24 h processed for further experiments.

Supernatants containing PFA-fixed LASV (30 ml) were again cleared twice by low-speed centrifugation (5,000 g for 30 min) to remove potential PFA-induced aggregates and subsequently pelleted through 20% sucrose cushion by ultracentrifugation (100,000 g for 2 h). Pellets were resuspended in 150 μl phosphate-buffered saline (PBS) for 12 h at 4°C. The samples were further purified by pelleting twice through 20% sucrose cushion by ultracentrifugation (100,000 g for 2 h) and resuspended in PBS. The sample was laid onto a 10%–60% sucrose gradient and centrifuged (100,000 g for 12 h). The gradient was fractionated using a gradient fractionator (Biocomp, Fredericton, NB, Canada) and fractions were collected using a fraction collector (Gilson, Middleton, WI) while monitoring the UV absorbance at 254 nm using a BioProbe (Biocomp, Fredericton, NB, Canada) together with EM-1 UV monitor (Bio-Rad Laboratories, Hercules, CA). Ten 100-μl fractions collected around the absorbance peak were pooled, pelleted and resuspended as described above.

Expression and purification of virus-like particles

Madin–Darby canine kidney (MDCK-II) cells stably expressing LASV GP protein [22] were cultured in DMEM supplemented with 10% FCS for 96 h. The medium was replaced by DMEM with 2% FCS for VLP expression. After 96 h, the cell supernatant was cleared twice by centrifugation (5,000 g for 30 min). Virus-like particles (VLPs) were pelleted through 20% sucrose cushion by ultracentrifugation (125,000 g for 3 h). Pellets were resuspended in 150 μl PBS for 12 h at 4°C.

Solutions of succinic acid, sodium dihydrogen phosphate and glycine were mixed in 2:7:7 molar ratio to make 50 mM SPG buffer. The buffer was adjusted to the desired pH by adding 0.5 M sodium hydroxide or 1 M hydrogen chloride. For Western blot analysis of particles at different pH, 300 μl of sample was incubated with 5 ml SPG buffer at the desired pH for 5 min at room temperature, then pelleted through sucrose cushion and resuspended as described above. Protein concentration of purified and different pH treated VLPs was measured using Nanodrop (Thermo Fischer Scientific).

Western blot analysis

An aliquot corresponding to 8 μg protein from each sample was separated by SDS-PAGE on a NuPAGE 4–12% Bis-Tris gel (Life Technologies, Thermo Fischer Scientific) and subsequently transferred to nitrocellulose membranes by using a dry-blotting system (iBlot; Life Technologies). Membranes were blocked in 5% milk in PBS buffer for 2 hours. Membranes were incubated with 1/3,000 diluted primary antibodies (monoclonal mouse antibody AC1 against GP-C and GP1 [kindly provided by Marie-Claude Georges-Courbot]; polyclonal rabbit antibody GP4 recognizing GP-C and GP2 [38]) for 1 h followed by an incubation with 1/1,000 diluted horseradish peroxidase- conjugated secondary antibodies against mouse and rabbit (Qiagen, Crawley, UK) for 1 h. The membrane was soaked in 5 ml of enhanced chemiluminescence reagent mixture (GE Healthcare, Buckinghamshire, UK) and visualized in a ChemiDoc MP gel imager (Bio-Rad Laboratories, Hercules, CA). A 1-μl aliquot of pre-stained protein maker (Benchmark; Life Technologies) was used to determine the protein mass.

Expression and purification of human LAMP1 membrane distal domain

A fragment of human LAMP1 (residues 27–194; UniProt accession no. P11279), predicted to correspond to the membrane-distal β-prism domain [29], was cloned into the pHLsec mammalian expression vector [39]. The LAMP1 domain was transiently expressed in HEK293T cells in the presence of the α-mannosidase inhibitor, kifunensine [40], with 2 mg of DNA transfected per litre of cell culture. Cell culture supernatant was collected 3 days post-transfection, filtered and diafiltrated against 10 mM Tris (pH 8.0) and 150mM NaCl. Purification comprised a two-step immobilized metal-affinity and size-exclusion procedure. Size-exclusion purification was carried out using a Superdex 200 10/30 column equilibrated against 10 mM Tris (pH 8.0) and 150 mM NaCl.

Binding of human LAMP1 membrane distal domain to VLPs

Enzyme-linked immunosorbent assays were performed in 96-well Nunc-Immuno PolySorp plates (Sigma) to which VLPs were bound overnight at a concentration of 10 μg/ml. Plates were blocked with PBS containing 5% milk powder and incubated with varying concentrations of purified LAMP1 membrane distal domain at different pH. LAMP1 detection was mediated using a rabbit derived anti-6His primary (Abcam, UK), and goat-anti rabbit-HRP (Abcam) antibody combination. Binding was assessed on the basis of optical absorbance at 405 nm after 30 min following HRP substrate addition.

Cryo-EM sample preparation

An aliquot (3 μl) of purified LASV or VLP sample was mixed with 3 μl of colloidal 6-nm gold particles coupled to bovine serum albumin (Aurion, Wageningen, The Netherlands) and applied on a plasma cleaned EM grids coated with holey carbon (C-flat; Protochips, Raleigh, NC). For low pH treatment of VLPs, the grid was floated for three minutes on top of a 2-ml droplet of SPG buffer at the desired pH. The grids were vitrified using a plunger device (CP3; Gatan, Pleasanton, CA) by blotting the sample for 3 s followed by plunge-freezing into a mixture of liquid ethane (37%) and propane (63%) [41].

Electron cryomicroscopy and tomography

All electron cryomicroscopy data were collected using a 300-kV transmission electron microscope (TF30 ‘Polara’; FEI, Eindhoven, Netherlands) operated at liquid nitrogen temperature. SerialEM [42] was used for acquiring low dose, single axis tilt series (from –45° to 45° with 5° angular sampling). A direct electron detector camera (K2 Summit; Gatan, Pleasanton, CA) mounted behind a post-column energy filter (QIF Quantum LS; Gatan, Pleasanton, CA) was used to acquire data at zero energy-loss mode (slit width 20 eV). At each tilt, a movie consisting of eight frames with total exposure time of 1.6 s was collected at a calibrated magnification of ×37,000 in electron counting super resolution mode, corresponding to a pixel size of 0.675 Å. Tilt series were recorded at nominal defocus values ranging from 2.0 to 3.5 μm under focus and the total electron dose was ~60 e^–/Ų.

Image pre-processing and tomogram generation

Drift correction [43] was used to correct for the electron beam induced motion by averaging eight frames for each tilt and applying 2× binning in Fourier space. IMOD package [44] was used to reconstruct three-dimensional tomograms from the stacks of tilted images. Images were aligned using the 6-nm gold beads as fiducial markers (S11 Fig). Prior to reconstruction, contribution from the gold beads was computationally removed, contrast transfer function (CTF) parameters were manually estimated (S12 Fig) and the effects of the CTF were corrected in the images by phase flipping [45]. For CTF background correction, noise images were recorded and processed the same was as actual tilt images. The amplitudes were not modified during CTF correction. Further 2× binning was applied, resulting in the final pixel size of 2.7 Å. LASV virions and VLPs were extracted from the full tomograms for further analysis.

Sub-tomogram averaging

Sub-tomogram alignment and averaging was carried out in Dynamo, which takes into account the missing wedge in tomographic data and allows parallel and graphics processing unit accelerated computations [46]. For initial template generation, extracted virion and VLP volumes were low-pass filtered to 80 Å using Bsoft [47]. Spikes were manually picked from the volumes of LASV and VLPs at pH 7 and pH 5 using Dynamo [46]. Dynamo tomoview program was used to create a surface model approximating the viral membrane, assuming either spherical or ellipsoidal geometry. Surface normals of the model surface provided initial orientations of the picked spikes. Spikes were extracted from the unfiltered tomograms into boxes of 128×128×128 voxels and averaged together. Cylindrical symmetry and low-pass filter to 35-Å resolution were applied on the average to reduce template bias.

The orientations were refined using the standard refinement strategy in Dynamo that implements an adaptive low-pass (‘push-back’) filter to further mitigate effects of template bias. Iterative refinement was performed in several stages (Supplementary Methods in S1 Text) [48–50]. Initially, no symmetry was applied to not bias the structure towards any particular symmetry. Only after three-fold appearance became evident in the asymmetric average (S13A Fig; C1), three-fold symmetry was applied in subsequent iterations (S13A Fig; C3). We further tested the effects of the starting model on the resulting structure. Even when the refinement was started with an initial model with wrong symmetry, it converged to a trimeric structure (S13B Fig). Typically a total 20 iterations were run until the resolution stopped improving (S13C Fig). In addition to the standard ‘push-back’ refinement, we carried out further tests with an alternative, so called ‘gold-standard’ refinement strategy using the VLP pH 7 data set. Both strategies yielded a trimeric spike at comparable resolutions (S14 Fig; Supplementary Methods in S1 Text).

To increase the size of the VLP pH 5 dataset and to locate all spikes in the VLP pH 3 data, we used template matching restricted on the VLP membrane surface. First, evenly distributed and oriented pseudo-particles (denoted here as ‘seeds’) were created on the surface of the particles [49,50] using Dynamo tomoview function using an ellipsoidal or a spherical surface model [46]. Seeds were defined with a seed-to-seed spacing of 24 pixels, resulting on average 430 and 340 seeds for pH 5 and pH 3 VLPs, respectively. As the seeds can correspond both to membrane areas that have spikes (true positives) and to areas that are devoid of spikes (false positives), these need to be differentiated. We thus used two templates in a multi-reference refinement run. First, a low-pass filtered cylindrically averaged spike, generated from the earlier VLP pH 5 average, served as a template for the true positives. Second, a spike-free membrane model served as a template for the false positives. The template that correlated the best determined whether the sub-tomogram in the given ‘seed’ position was a true or false positive. Shifts of 20 pixels were allowed lateral to the membrane. Unique spike sub-tomograms were identified by removing seeds that were too close to another seed. The included sub-tomograms were then subjected to structure refinement similar to the manually picked spikes. No spikes selected for the refinement were excluded after this stage.

The positions of the seeds were then iteratively refined, similarly as described above for manually picked spikes. Low-pass filtered cylindrical average of the densities extracted at seed positions was used as an initial template. The seeds were allowed mainly to shift only in the direction normal to the membrane. A smoothness criterion was implemented as a custom Dynamo function to weight these shifts by the shifts of the neighboring seeds. Similarly to motion correction of particles in single particle processing [51], the weight of each neighbor was adjusted by Gaussian function taking into account the distance to the seed in question, the neighbors farther away from the seed contributing less than the neighbors closer to the seed.

The resolution of the final maps was estimated by FSC, by splitting the sub-tomograms into two half datasets and using 0.5 as threshold [52]. As sub-tomograms extracted from the same virion (or VLP) may not be fully independent, each virion (or VLP) contributed sub-tomograms to one of the half datasets, but not both.

Structure analysis

To putatively assign densities to different structural components, the averaged density maps were computationally segmented using Segger [53] in UCSF Chimera [54]. The isosurface threshold in the spike averages was set to match the expected volume of the spike ectodomain (290,000 ų), calculated from the estimated spike ectodomain trimer mass (230 kDa, including glycosylations) assuming density of 0.81 Da / ų. For the spike at pH 3 lagging GP1, the volume was matched to correspond to three copies of GP2 (86 kDa; 106,000 ų). Spike ectodomain densities were then segmented. The volume corresponding to the LASV GP intra-viral segments was measured in UCSF Chimera.

To create a composite model for the LASV virion, sub-tomogram refinement parameters were first converted from Dynamo tables to a STAR file compatible with Jsubtomo[50]. The segmented densities for the spike protomers, membrane and matrix layer were then placed on their original places relative to the virion by using jsubtomo_create_model.py. An isosurface representation, also showing the RNP density inside of the virion was rendered in UCSF Chimera.

Fitting of atomic coordinates

We fitted the atomic coordinates of LASV GP1 (PDB:4ZJF) [10] to our VLP pH 5 spike using Segger [53]. Each segment assigned to a GP subunit was split into four smaller segments (S10A and S10B Fig). Each of the four chains present in the GP1 crystal structure were fitted to the four GP segments. From the total of 1,000 evenly rotated fits, unique fits were created by further optimization and by clustering fits that were less than 5.0 Å and 3.0 degrees apart (S10C Fig).